1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.

Subculturing

1. Centrifuge for 5 minutes 300 xg and discard culture medium.2. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.3. Add appropriate aliquots of the cell suspension to new culture vessels.Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:8 is recommended Medium Renewal: 2 to 3 times per week

Mycoplasma

Mycoplasma Status: Negative (MycoAlert Kit)

Freeze Medium

Complete growth medium 90%; DMSO, 10%

Safety Considerations

The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.

Ship

Dry ice

Product Purity

>98.0% as determined by Analysis by RP-HPLC & analysis by SDS-PAGE.

Product Type

Humanized (from mouse) IgG2

Product Storage

It should be stored in liquid or vapor phase nitrogen. Reconstituted antibody aliquots should avoid repeated freeze-thaw cycles.

Background

Introduction

Visilizumab is a humanized monoclonal antibody and is being investigated for use as an immunosuppressive drug in patients with ulcerative colitis and Crohn's disease.

Antigen Description

The protein encoded by this gene is the CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The genes encoding the epsilon, gamma and delta polypeptides are located in the same cluster on chromosome 11. The epsilon polypeptide plays an essential role in T-cell development. Defects in this gene cause immunodeficiency. This gene has also been linked to a susceptibility to type I diabetes in women.

Involvement in Disease

Visilizumab is being investigated for use as an immunosuppressive drug in patients with ulcerative colitis and Crohn's disease.